Dynamin, actin and microtubules: cytoskeletal crosstalk in podocytes

项目摘要/摘要 慢性肾脏疾病的全球流行正在以惊人的速度发展。仅在美国， 肾小球疾病影响了大约2000万人，这一数字在过去两年中大约翻了一番。 几十年。与肾脏有关的疾病正在迅速避开目前可用的治疗选择和资源。 足细胞是肾小球内独特的细胞，它有一个复杂的组织，由 细胞体、微管驱动的膜延伸(初级过程)和肌动蛋白驱动的细胞骨架 膜延伸(足突)。由于足细胞的细胞骨架由三个不同的 元素--肌动蛋白细丝、微管和中间细丝--它们的细胞骨架必须协调一致 调节足细胞完成其复杂的细胞功能。细胞骨架元素之间的配位是 通过信号通路实现，这涉及到常见的调控因子，如Rho GTP酶以及 大的GTP酶动力蛋白。我们已经证明，动力素直接调节足细胞中的肌动蛋白细胞骨架。 我们的新发现表明，动力蛋白也调节微管--特别是动力蛋白形成螺旋 围绕着微管。动力蛋白螺旋显示出非常高的GTP水解率，这反过来又抑制了 通过降低GTP的局部浓度进行微管聚合。需要较高的GTP浓度 用于高效的微管蛋白齐聚，因为微管蛋白使用GTP结合来引发其聚合 微管。此外，动力蛋白螺旋对GTP的高水解率也使微管收缩为 小半径，从而破坏微管的稳定，并引发微管灾难。因此，这两位 GTP酶，动力蛋白和微管蛋白，受其附近的GTP的可用性和动力蛋白的能力调节。 在微管周围形成螺旋状的寡聚。动力素直接影响微管动力学的能力 肌动蛋白提示，动力素可能是足细胞中这两种细胞骨架蛋白的一种。 用于相互通信。在特定的目标1中，我们使用实时单分子成像来解密 动力蛋白-微管和动力蛋白-肌动蛋白相互作用的机制细节。在《特定目标2》中，我们使用了一组 不同的动力蛋白突变体、足细胞培养、使用纯化蛋白的生物化学和电子显微镜 研究动力蛋白在足细胞微管调节中的作用，以及微管如何影响足细胞。 局灶性粘连和应力纤维的形成。在具体目标3中，我们通过在动物身上使用 我们新的动力素特异性小鼠模型，以及HIV相关肾病(HIVAN)的小鼠模型。我们 测试有寡聚倾向并有望促进微管形成的动力蛋白突变体 解聚可以通过使用促进或抑制足细胞损伤的微管药物来抵消 微管聚合。鉴于微管在胞质分裂中的重要作用，我们还将研究 微管药物对HIVAN足细胞增殖的影响。

Project Summary/Abstract The global epidemic of chronic kidney disease is progressing at an alarming rate. In the United States alone, glomerular kidney diseases affect some 20 million people, and this number has roughly doubled in the last two decades. Kidney-related diseases are rapidly eluding current available treatment options and resources. Podocytes are unique cells within the kidney glomerulus, which have a complex organization consisting of a cell body, microtubule-driven membrane extensions (primary processes), and actin-cytoskeleton driven membrane extensions (foot processes). Since the podocyte cytoskeleton is composed of three distinct elements - actin filaments, microtubules, and intermediate filaments - their cytoskeleton must be coordinately regulated for podocytes to fulfill their complex cellular function. Coordination between cytoskeletal elements is achieved through signaling pathways, which involve common regulators such as Rho GTPases as well as the large GTPase dynamin. We have shown that dynamin directly regulates the actin cytoskeleton in podocytes. Our novel findings suggest that dynamin also regulates microtubules – specifically that dynamin forms helices around microtubules. Dynamin helices exhibit very high rates of GTP hydrolysis, which in turn inhibit microtubule polymerization by lowering the local concentration of GTP. A high GTP concentration is required for efficient tubulin oligomerization since tubulin uses GTP binding to initiate its polymerization into microtubules. Additionally, high rate of GTP hydrolysis by dynamin helices also constricts microtubules to a small radii, thus destabilizing microtubules and initiating microtubule catastrophe. Therefore, these two GTPases, dynamin and tubulin, are regulated by the availability of GTP in their vicinity and dynamin's ability to oligomerize into helices around microtubules. The ability of dynamin to directly affect dynamics of microtubules and actin suggests that dynamin might be one of the proteins in podocytes that these two cytoskeletal proteins use to communicate with each other. In Specific Aim 1 we use real time single-molecule imaging to decipher mechanistic details of dynamin-microtubule and dynamin-actin interactions. In Specific Aim 2 we use a panel of diverse dynamin mutants, podocyte cell culture, biochemistry using purified proteins, and electron microscopy to investigate the role that dynamin plays in regulating microtubules in podocytes, and how microtubules affect the formation of focal adhesions and stress fibers. In Specific Aim 3 we test our hypothesis in animals by using our novel dynamin-specific mouse model, and a mouse model of HIV-associated nephropathy (HIVAN). We test whether a dynamin mutant that has the propensity to oligomerize and is expected to promote microtubule depolymerization can counteract podocyte injury by using microtubule drugs that either promote or inhibit microtubule polymerization. Given the essential role of microtubules in cytokinesis, we will also examine the effect of microtubule drugs on podocyte proliferation observed in HIVAN.